The effects of aging on pre-prepared and post-prepared basic fuchsin dyes used in the Feulgen nucleal reaction, for quantitative cytophotometric measurements

Abstract

The objective of this investigation was to determine the effects of the aging of pre-prepared (hydrated) Basic Fuchsin on the quantitative cytophotometric properties of the Feulgen nucleal reaction. This was done by comparing six separate preparations of Schiff's reagent prepared from Basic Fuchsins manufactured in 1972, 1974, 1976, 1985 and 1987 respectively.

Changes in reactivity of post-prepared Schiff's reagents over time were analyzed in order to determine the stability, and thus the effect of dye ageing on quantitative determinations of cellular DNA content. The establishment of the time period (duration) for stability is important as part of determination of reaction parameters best suited to producing optimal Feulgen intensities.

To accomplish these objective the two wavelength methods of quantitative microspectrophotometry was utilized (Patau 1952). Measurements were made using myxamoebae and plasmodia of the slime mold "Didymium iridis," since both haploid and diploid cells are known to regularly occur in this organism.

The analysis of nuclear DNA is illustrated as histograms for each stain (number of nuclei vs. relative dye concentration of DNA). The mean values, standard deviations and standard errors are also recorded.

Upon analysis of the data obtained it was determined that the shelf-life of the undissolved stain over a long length of time does not affect its quantitative staining ability, as long as the dyes are properly stored unhydrated and handled properly. The intensity of the dye concerning nuclear uptake and subsequent stoichiometric color change developed appears to be initially influenced by the chemical composition of the stain rather than the age of the unhydrated original dye. Basic Fuchsins with CI number of 42500 have high initial affinity for DNA than stains having CI number 42510 according to the measured DNA content.

Post-prepared hydrated dye can be used safely for at least two weeks, and give optimal quantitative results during this period of time. Following this time, however the staining intensity of the nuclei produced by the dye is gradually diminished. However, the stain is still capable of stoichiometric DNA measurements in arbitrary units.